One of the major goals of both biotechnology and nanotechnology (1,2) is the assembly of novel biomaterials that can be used for analytical, industrial or therapeutic purposes. A particular aim is to construct individual objects and devices on the nanometer scale, utilizing the informational macromolecules, e.g. polypeptides and polynucleotides, of biological systems.
Nature provides many examples of elegant polypeptide or polynucleotide constructs on a nanometer scale. For example, the type of subcellular organelle known as a ribosome is a sophisticated machine constructed of polynucleotides and polypeptides. As is well known to the art, a ribosome functions in a machine-like manner in order to "read" the genetic sequence coded by a messenger ribonucleic acid (RNA) strand and to generate the corresponding polypeptide. The ability to construct functional artificial components on a nanometer scale from polypeptides and/or nucleic acids would provide the capability of creating artificial tools and reagents able to mimic the function of natural subcellular organelles and to perform other useful functions, not necessarily present in the natural state, for diagnostic, therapeutic or industrial purposes. For example, such a construct would provide a useful 3-dimensional scaffolding upon which enzymatic or antibody binding domains may be linked to provide high density multivalent processing sites to link to and solubilize otherwise insoluble enzymes, or to entrap, protect and deliver a variety of molecular species, and the like.
Polypeptides play a variety of prominent functional roles in living cells, including enzymatic, regulatory and structural activities; hence, substantial effort has gone into the engineering of polypeptides (e.g., 3-5). However, less attention has been paid to the structural possibilities of nucleic acids. The stable form of naturally-occurring DNA is a linear double helical molecule (6), with limited potential for the construction of complex objects.
During the past several years, a number of investigations of the physical properties of deoxyribonucleic acid (DNA) branched junctions (10, 19-29), as well as their susceptibility to resolving enzymes (26, 30-32) have been reported. These studies have been stimulated by the role of branched DNA molecules as intermediates in the process of genetic recombination (33). In addition, the possibility of using branched DNA molecules to construct nanometer scale (also referred to herein as "nanoscale") objects has been explored. In prior work a series of macrocycles (cyclic trimers, tetramers, etc.) was formed by oligomerizing 3-arm junctions (34) or 4-arm junctions (35) containing a pair of complementary cohesive ends. The presence of numerous closed products from those ligations indicates a large degree of flexibility in the angles between arms (over long ligation times), regardless of how well-defined the structure of an individual junction may appear to be (27-29).
Each of these previous studies has produced a closed object that may be described as 2-connected (37). Although sharp kinks are introduced into the constructs by the presence of the junctions, the closed figures formed are essentially cyclic flexed variations on a linear theme. In addition, the earlier constructions are not inherently 3-dimensional molecules, although they may happen to be non-planar. Thus, it can be readily appreciated that provisions of a 3-connected or closed covalent three dimensional structure of oligonucleotides, and of methods of making and using such structures, would be a highly desirable advance over the current state of technology.